The invention relates to antibodies and methods of using antibodies to treat disorders associated with CXCR3 signaling such as diabetes mellitus type 1 (type I diabetes; T1D).
Diabetes is characterized by chronic hyperglycemia resulting from a lack of insulin action, along with various characteristic metabolic abnormalities. Diabetes can be broadly divided into type I and type II. T1D is characterized by the loss of pancreatic β-cells of the Langerhans' islets, while type II diabetes is characterized by reductions in both insulin secretion and insulin sensitivity (insulin resistance). In the United States, the prevalence of diabetes is about 2 to 4 percent of the population, with type I diabetes (also known as insulin-dependent or IDDM) making up about 7 to 10 percent of all cases.
Type I diabetes is characterized by the failure to produce sufficient insulin to maintain glucose homeostasis. This disorder is believed to be caused by autoimmune-mediated destruction of the pancreatic β-cells. Autoimmunity associated with type I diabetes involves the participation of both B and T autoreactive lymphocytes. Indeed, up to 98% of type I diabetes patients have antibodies against one or more of their own β-cell antigens, including insulin, glutamic acid decarboxylase (GAD), insulinoma antigen-2 and insulinoma antigen-2b (IA-2 and IA-2 β), and heterogeneous islet cell cytoplasmic antigens (ICAs). Although it may not always be determinative, the level of one or more autoantibodies generally correlates with the state of β-cell destruction. Irvine, et al., Diabetes, 26:138-47 (1997); Riley, et al., N. Engl. J. Med., 323:1167-72 (1990). Accordingly, autoantibodies can serve as indicators of the development of autoimmune diabetes and together with metabolic changes can predict the risk of developing diabetes in relatives of T1D patients
The development of type I diabetes may be mediated by autoreactive T cells, as evidenced by tissue biopsies obtained near the time of T1D diagnosis that show the islets infiltrated with activated T cells. Bottazzo et al., N. Engl. J. Med., 313:353-60 (1985); Hanninen et al., J. Clin. Invest., 90:1901-10 (1992); Itoh et al., J. Clin. Invest., 92:2313-22 (1993); Imagawa, et al., Diabetes, 50:1269-73 (2001).
Chemokine (C-X-C motif) receptor 3 (CXCR3), also known as G protein-coupled receptor 9 (GPR9), CD183, IP-10 receptor, and Mig receptor, is a chemokine receptor expressed on autoreactive T cells that have been implicated in a range of physiological processes and related disorders, such as T1D. CXCR3 is largely absent from naïve T cells but is upregulated upon activation with antigen and recruits activated cells to sites of tissue inflammation in response to its primary ligands: CXCL9, CXCL10, and CXCL11. β cells have been shown to predominately express CXCL10, with lower levels of CXCL9, in mouse models of T1D (Christen et al The Journal of Immunology, 2003, 171: 6838-6845; Morimoto et al. J Immunol 2004; 173; 7017-7024; Sarkar et al. Diabetes. 2012 February; 61(2):436-46); and in islets from T1D patients having insulitis (Uno et al 2010; Roep et al Clinical and Experimental Immunology, 2003, 159: 338-343; Sarkar et al. Diabetes. 2012 February; 61(2):436-46). In addition, T cells that have infiltrated the pancreas have been shown to express CXCR3 in T1D mice models and type 1 diabetes patient pancreas samples (Christen et al, The Journal of Immunology, 2003, 171: 6838-6845; Van Halteren et al., Diabetologia 48:75-82 (2005); Uno et al 2010; Roep et al., Clinical and Experimental Immunology, 2003, 159: 338-343; Sarkar et al., Diabetes. 2012 February; 61(2):436-46). Furthermore, knockout mice deficient in CXCR3 demonstrate a significant delay in onset and a reduction in incidence of T1D (Frigerio et al., Nature Medicine 8:1414-1420 (2002)), while overexpression of CXCL10 in the islets of transgenic mice promotes T cell infiltration and accelerates the onset of T1D (Rhode et al., J. Immunol. 175(6): 3516-24 (2005)). Neutralization of CXCL10 by antibody treatment has been shown to be protective (Christen et al., The Journal of Immunology, 2003, 171: 6838-6845).
There are three isoforms of CXCR3, denoted A, B, and Alt., that have been identified in humans (Lasagni et al. J. Exp. Med. 2003 197:1537; Ehlert et al J. Immunol. 2004; 173; 6234-6240), CXCR3-A binds to the CXC chemokines CXCL9 (MIG), CXCL10 (IP-10), and CXCL11 (I-TAC); CXCR3-B also binds to these targets but also binds CXCL4; CXCR3-Alt appears to interact with CXCL11. Although alternative splicing leads to the generation of several protein isoforms of CXCR3, CXCR3-A is the predominant form in vivo as the CXCR3-B and CXCR3-Alt are expressed at much lower levels at the protein levels. Lasagni et al. J. Exp. Med. 2003 197:1537; Ehlert et al J. Immunol. 2004; 173; 6234-6240.
Efforts to disrupt the CXCR3 pathway using small molecule inhibitors of CXCR3 have not proved fully effective. Christen et al., Clin Exp. Immunol. 165: 318-328 (2011). Accordingly, research has focused on antibodies and other methods of disrupting CXCL10, primarily before the onset of diabetes. Morimoto et al., J. Immun. 173: 7017-7024 (2004); Oikawa et al., Rev. Diabet. Stud. 7: 209-224 (2010).
In view of the prevalence of T1D and other disorders in which CXCR3 has been implicated, a need exists for additional methods that target CXCR3 signaling, e.g., to treat or reduce the progression of a disorder such as T1D in a patient.
Disclosed herein are antibodies and methods of using antibodies that are capable of binding to CXCR3. In some embodiments, the antibodies can be used to prevent, treat or reduce the early progression of T1D in a subject by targeting the CXCR3 pathway. The antibodies and methods rely, at least in part, on the surprising result that neutralizing antibodies directed to CXCR3 can prevent onset of T1D in NOD mice when administered prior to disease onset, or can reverse the course of disease when administered in the new-onset stage of T1D in NOD mice. Furthermore, neutralization of CXCR3 activity is not associated with a significant impairment of the normal operation of the patient's immune system, thereby reducing the undesirable side effects of antibody therapy.
Accordingly, in one aspect, disclosed herein are antibodies and antigen binding fragments capable of neutralizing the activity of CXCR3. In certain embodiments, CXCR3 neutralizing antibodies may be characterized by the ability to bind to a peptide selected from residues 1-58, 1-16, or 1-37 of SEQ ID NO:1. In some embodiments, the antibodies comprise all or portions of antibody clones (Cl) designated Cl 12, Cl 135, Cl 82, Cl 53 and/or Cl 4. In certain embodiments, variants of antibodies Cl 12, Cl 135, Cl 82, Cl 53 and/or Cl 4 are provided, including CDR-grafted, humanized, back mutated, and fully human variants of the disclosed antibodies. In particular embodiments, the antibody comprises one or more complementarity determining regions (CDRs), e.g., one or more of heavy chain CDR1, CDR2, and CDR3, and/or one or more of light chain CDR1, CDR2, and CDR3, from clones Cl 12, Cl 135, Cl 82, Cl 53 and/or Cl 4 or any of the variants of clones 4, 12, 53, 82, and 135 disclosed herein. In some embodiments, the antibodies from Cl 12, Cl 135, Cl 82, Cl 53 and/or Cl 4, or the chimeric and humanized versions thereof, exhibit certain beneficial properties, as compared to anti-CXCR3 clones 5H7, 7H5, V44D7, 1C6, and/or 49801. For example, the antibodies disclosed herein can exhibit increased binding affinity as compared to the anti-hCXCR3 clones 5H7, 7H5, V44D7, 1C6, and 49801. For example, the antibody may exhibit 1, 2, 3, 4, 5, or more fold better affinity (or any value in between) over anti-CXCR3 antibodies such as 1C6, e.g., as measured by surface Plasmon resonance (e.g., using a BIACORE™ assay). The humanized antibodies disclosed herein also have a predicted reduction in immunogenicity as compared to the mouse anti-hCXCR3 clones 5H7, 7H5, V44D7, 1C6, and 49801. In addition, heavy chain clones 4.7-4.11 disclosed herein have been optimized to remove a deamidation site at positions 58 and 59 (using IMGT numbering) and thereby enhance stability over the initial mouse anti-hCXCR3 heavy chain variable domain (VH) CDR2 sequence.
In another aspect, the present disclosure provides methods of prophylaxis prior to T1D onset, as well as methods of treating or reducing the progression of new onset T1D in a subject by administering an effective amount of a CXCR3 neutralizing antibody. In particular embodiments, the subject is a mammal, such as a human.
In certain embodiments, the subject having new onset T1D is treated by the methods disclosed herein within 6 months of clinical diagnosis. In other embodiments, the subject is treated more than 6 months after clinical diagnosis, wherein the subject retains residual fasting integrated serum C-peptide levels of at least about 0.2 nmol/L.
In some embodiments, subjects may be characterized by elevated fasting blood glucose levels in the absence of exogenous insulin above 120 mg/dL or an abnormally low fasting integrated serum C-peptide level of about 0.033 to 1.0 nmol/L×min during C-peptide stimulation. In particular embodiments the CXCR3 neutralizing antibody is administered at a dose of about 0.03-3.7 mg/kg/dose. In some embodiments, the subject is administered at least one dose of antibody. In certain embodiments, the subject is administered repeat doses of antibody (e.g., at least yearly, quarterly, bimonthly, monthly, biweekly, weekly, or daily). In further embodiments, the methods described above may further comprise the step of administering an immunosuppressant and/or β-cell stimulating agent concurrently or sequentially (before or after) administering the CXCR3 neutralizing antibody.
In various embodiments, the anti-CXCR3 antibodies disclosed herein are administered to treat a condition characterized by abnormal CXCR3 expression. In some embodiments, the anti-CXCR3 antibodies are administered to treat any condition that can benefit from the downregulation and/or neutralization of CXCR3 activity. In some embodiments, the anti-CXCR3 antibodies disclosed herein are administered to treat T1D.
Additional embodiments and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The embodiments and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment(s) of the invention and together with the description, serve to explain the principles of the invention.